The major goal of this project is to take advantage of the recent advances made in the field of 31p and 13C nuclear magnetic resonances (NMR) spectroscopy to study the energetics and metabolism of perfused rat hearts under different conditions of substrate supply and work. Emphasis will be given to the use of 13C-enriched substrates to study regulation of the citric acid cycle, its interactions with the malate-asparate cycle and mechanisms for increasing or decreasing net pool sizes of citric acid cycle intermediates. 13C NMR spectra will be obtained using both intact hearts and perchloric acid extracts. The latter will also be used for enzymatic assay of metabolites to aid identification and quantitation of resonances obtained from individual 13C-labeled carbon atoms from the 13C spectra. The information provided from 13C specific labeling patterns of intermediates, together with knowledge of their total pool sizes as determined by metabolic analyses, will be used to construct a flux model of the citric acid cycle and interrelated transamination steps. The model will be used to test various hypotheses concerning regulation of flux in the citric acid cycle under different conditions of substrate supply and work output of the heart during the steady state as well as transition states when cycle flux is nonuniform. The following major specific aims will be addressed. 1. Identification of rate-controlling steps in the citric acid cycle and assessment of enzyme control strengths by inhibitor titration studies. 2. Assessment of the existence and metabolic significance of metabolite compartmention and substrate channeling. 3. Effects of varying flux rates through the malate-asparate cycle on the regulation of flux through citrate syntase, Alpha-ketoglutarate dehydrogenase and asparate aminotransferase. 4. Investigation of the mechanisms responsible for causing net changes of the tissue contents of citric acid cycle intermediates. 5. Effects of acetoacetate in the absence and presence of propionate on flux through succinate thiokinase and regulation of the citric acid cycle from Alpha-keto-glutarate to malate. 6. Investigation of citric acid cycle function in reoxygenated post-ischemic hearts.